Expression and accurate processing of yeast penta-ubiquitin in Escherichia coli.

An expression vector (pSJyub-5) was constructed which contained five repeats of the "yeast ubiquitin gene" regulated by a heat-inducible lambda PL promoter. The vector, when expressed in Escherichia coli, produced a penta-ubiquitin of approximately 42 kDa. Purified penta-ubiquitin was found to be as active as the human mono-ubiquitin in the in vitro ATP/ubiquitin-dependent proteolytic assay of the reticulocyte lysate, indicating that the expressed gene product was recognized by the enzymes involved in the ATP/ubiquitin-dependent proteolytic pathway.

Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin.

To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified.

Flux regulation in glycogen-induced oscillatory glycolysis in cell-free extracts of Saccharomyces carlsbergensis.

To localise the controlling point of the glycolytic system, the temporal changes in concentrations of glycolytic intermediates have been analysed after addition of glycogen to a substrate-depleted yeast extract. Three sequential metabolic states are clearly observable: a transition state at which there is continuous accumulation of the intermediates before the glyceraldehydephosphate dehydrogenase (GAPDH, EC 1.2.